Glucose-6-Phosphate Dehydrogenase from the Human Pathogen Trypanosoma cruzi Evolved Unique Structural Features to Support Efficient Product Formation.

Glucose-6-phosphate dehydrogenase (G6PDH) is the key enzyme supplying reducing power (NADPH) to the cells, by oxidation of glucose-6-phosphate (G6P), and in the process providing a precursor of ribose-5-phosphate. G6PDH is also a virulence factor of pathogenic trypanosomatid parasites. To uncover the biochemical and structural features that distinguish TcG6PDH from its human homolog, we have solved and analyzed the crystal structures of the G6PDH from Trypanosoma cruzi (TcG6PDH), alone and in complex with G6P. TcG6PDH crystallized as a tetramer and enzymatic assays further indicated that the tetramer is the active form in the parasite, in contrast to human G6PDH, which displays higher activity as a dimer. This quaternary structure was shown to be particularly stable. The molecular reasons behind this disparity were unveiled by structural analyses: a TcG6PDH-specific residue, R323, is located at the dimer-dimer interface, critically contributing with two salt bridges per subunit that are absent in the human enzyme. This explains why TcG6PDH dimerization impaired enzyme activity. The parasite protein is also distinct in displaying a 37-amino-acid extension at the N-terminus, which comprises the non-conserved C8 and C34 involved in the covalent linkage of two neighboring protomers. In addition, a cysteine triad (C53, C94 and C135) specific of Kinetoplastid G6PDHs proved critical for stabilization of TcG6PDH active site. Based on the structural and biochemical data, we posit that the N-terminal region and the catalytic site are highly dynamic. The unique structural features of TcG6PDH pave the way toward the design of efficacious and highly specific anti-trypanosomal drugs.

Allosteric activation of bacterial response regulators: the role of the cognate histidine kinase beyond phosphorylation.

UNLABELLED: Response regulators are proteins that undergo transient phosphorylation, connecting specific signals to adaptive responses. Remarkably, the molecular mechanism of response regulator activation remains elusive, largely because of the scarcity of structural data on multidomain response regulators and histidine kinase/response regulator complexes. We now address this question by using a combination of crystallographic data and functional analyses in vitro and in vivo, studying DesR and its cognate sensor kinase DesK, a two-component system that controls membrane fluidity in Bacillus subtilis. We establish that phosphorylation of the receiver domain of DesR is allosterically coupled to two distinct exposed surfaces of the protein, controlling noncanonical dimerization/tetramerization, cooperative activation, and DesK binding. One of these surfaces is critical for both homodimerization- and kinase-triggered allosteric activations. Moreover, DesK induces a phosphorylation-independent activation of DesR in vivo, uncovering a novel and stringent level of specificity among kinases and regulators. Our results support a model that helps to explain how response regulators restrict phosphorylation by small-molecule phosphoryl donors, as well as cross talk with noncognate sensors. IMPORTANCE: The ability to sense and respond to environmental variations is an essential property for cell survival. Two-component systems mediate key signaling pathways that allow bacteria to integrate extra- or intracellular signals. Here we focus on the DesK/DesR system, which acts as a molecular thermometer in B. subtilis, regulating the cell membrane's fluidity. Using a combination of complementary approaches, including determination of the crystal structures of active and inactive forms of the response regulator DesR, we unveil novel molecular mechanisms of DesR's activation switch. In particular, we show that the association of the cognate histidine kinase DesK triggers DesR activation beyond the transfer of the phosphoryl group. On the basis of sequence and structural analyses of other two-component systems, this activation mechanism appears to be used in a wide range of sensory systems, contributing a further level of specificity control among different signaling pathways.

HemR is an OmpR/PhoB-like response regulator from Leptospira, which simultaneously effects transcriptional activation and repression of key haem metabolism genes.

Several Leptospira species cause leptospirosis, the most extended zoonosis worldwide. In bacteria, two-component systems constitute key signalling pathways, some of which are involved in pathogenesis. The physiological roles of two-component systems in Leptospira are largely unknown, despite identifying several dozens within their genomes. Biochemical confirmation of an operative phosphorelaying two-component system has been obtained so far only for the Hklep/Rrlep pair. It is known that hklep/rrlep knockout strains of Leptospira biflexa result in haem auxotrophy, although their de novo biosynthesis machinery remains fully functional. Haem is essential for Leptospira, but information about Hklep/Rrlep effector function(s) and target(s) is still lacking. We are now reporting a thorough molecular characterization of this system, which we rename HemK/HemR. The DNA HemR-binding motif was determined, and found within the genomes of saprophyte and pathogenic Leptospira. In this way, putative HemR-regulated genes were pinpointed, including haem catabolism-related (hmuO - haem oxygenase) and biosynthesis-related (the hemA/C/D/B/L/E/N/G operon). Specific HemR binding to these two promoters was quantified, and a dual function was observed in vivo, inversely repressing the hmuO, while activating the hemA operon transcription. The crystal structure of HemR receiver domain was determined, leading to a mechanistic model for its dual regulatory role.

Sevoflurane anesthesia deteriorates pulmonary surfactant promoting alveolar collapse in male Sprague-Dawley rats.

General anesthesia is frequently associated to transient hypoxemia and lung atelectasis. Although volatile anesthetics are safe and widely used, their potential role on anesthesia-induced pulmonary impairment has not been fully explored. In this study, we investigated the effect of volatile anesthetic sevoflurane on pulmonary surfactant composition and structure that could contribute to atelectasis. After 30 min of sevoflurane anesthesia, Sprague-Dawley rats showed increased levels of lyso-phosphatidylcholine and decreased levels of phosphatidylcholine associated with significant impairment in lung mechanics and alveolar collapse, but showed no deterioration of alveolar fluid reabsorption when compared to control group of rats anesthetized with pentobarbital. Exposure to sevoflurane altered the thermotropic profile of surfactant model membranes, as detected by fluorescence anisotropy. In this sense, sevoflurane-promoted fluidification of condensed phases could potentially impair the ability of surfactant films to sustain the lowest surface tensions. In conclusion, the observed changes in surfactant composition and viscosity properties suggest a direct effect of sevoflurane on surfactant function, a factor potentially involved in anesthetic-induced alterations in lung mechanics.

HPLC separation of human serum albumin isoforms based on their isoelectric points.

Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA-SHg(+)), HSA with Cys34 oxidized to sulfenic acid (HSA-SOH) and HSA oxidized to sulfinate anion (HSA-SO2(-)) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3-585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA.

Modulation of the reactivity of the thiol of human serum albumin and its sulfenic derivative by fatty acids.

The single cysteine residue of human serum albumin (HSA-SH) is the most abundant plasma thiol. HSA transports fatty acids (FA), a cargo that increases under conditions of diabetes, exercise or adrenergic stimulation. The stearic acid-HSA (5/1) complex reacted sixfold faster than FA-free HSA at pH 7.4 with the disulfide 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and twofold faster with hydrogen peroxide and peroxynitrite. The apparent pK(a) of HSA-SH decreased from 7.9±0.1 to 7.4±0.1. Exposure to H(2)O(2) (2mM, 5min, 37°C) yielded 0.29±0.04mol of sulfenic acid (HSA-SOH) per mole of FA-bound HSA. The reactivity of HSA-SOH with low molecular weight thiols increased ∼threefold in the presence of FA. The enhanced reactivity of the albumin thiol at neutral pH upon FA binding can be rationalized by considering that the corresponding conformational changes that increase thiol exposure both increase the availability of the thiolate due to a lower apparent pK(a) and also loosen steric constraints for reactions. Since situations that increase circulating FA are associated with oxidative stress, this increased reactivity of HSA-SH could assist in oxidant removal.

Expression, crystallization and preliminary X-ray crystallographic analysis of glucose-6-phosphate dehydrogenase from the human pathogen Trypanosoma cruzi in complex with substrate.

An N-terminally truncated version of the enzyme glucose-6-phosphate dehydrogenase from Trypanosoma cruzi lacking the first 37 residues was crystallized both in its apo form and in a binary complex with glucose 6-phosphate. The crystals both belonged to space group P2(1) and diffracted to 2.85 and 3.35 Å resolution, respectively. Self-rotation function maps were consistent with point group 222. The structure was solved by molecular replacement, confirming a tetrameric quaternary structure.

Genetic toxicology and preliminary in vivo studies of nitric oxide donor tocopherol analogs as potential new class of antiatherogenic agents.

Nitric oxide donor tocopherol analogs were found to be incorporated in low-density lipoprotein to release nitric oxide into the hydrophobic core of the lipoprotein, thus inhibiting lipid oxidation processes associated with atheroma plaque formation. Previously, we studied their cytotoxicity against human and murine macrophages as first selection for in vivo studies. Herein, we examined both the in vitro mutagenic and DNA-damage effects of selected compounds to further evaluate drug potential. While the compounds of interest were nongenotoxics in both experimental tests (Ames and alkaline comet), one of the potential blood metabolites exhibited genotoxicity (alkaline comet test), and the furazan derivative was mutagenic (Ames test). Two selected (nitrooxy and furoxan) compounds were studied in long- and short-term in vivo treatment, and in these conditions, animal toxicity was not evidenced, suggesting the possibility of these compounds as potential antiatherogenic drugs.

Factors affecting protein thiol reactivity and specificity in peroxide reduction.

Protein thiol reactivity generally involves the nucleophilic attack of the thiolate on an electrophile. A low pK(a) means higher availability of the thiolate at neutral pH but often a lower nucleophilicity. Protein structural factors contribute to increasing the reactivity of the thiol in very specific reactions, but these factors do not provide an indiscriminate augmentation in general reactivity. Notably, reduction of hydroperoxides by the catalytic cysteine of peroxiredoxins can achieve extraordinary reaction rates relative to free cysteine. The discussion of this catalytic efficiency has centered in the stabilization of the thiolate as a way to increase nucleophilicity. Such stabilization originates from electrostatic and polar interactions of the catalytic cysteine with the protein environment. We propose that the set of interactions is better described as a means of stabilizing the anionic transition state of the reaction. The enhanced acidity of the critical cysteine is concurrent but not the cause of catalytic efficiency. Protein stabilization of the transition state is achieved by (a) a relatively static charge distribution around the cysteine that includes a conserved arginine and the N-terminus of an α-helix providing a cationic environment that stabilizes the reacting thiolate, the transition state, and also the anionic leaving group; (b) a dynamic set of polar interactions that stabilize the thiolate in the resting enzyme and contribute to restraining its reactivity in the absence of substrate; but upon peroxide binding these active/binding site groups switch interactions from thiolate to peroxide oxygens, simultaneously increasing the nucleophilicity of the attacking sulfur and facilitating the correct positioning of the substrate. The switching of polar interaction provides further acceleration and, importantly, confers specificity to the thiol reactivity. The extraordinary thiol reactivity and specificity toward H(2)O(2) combined with their ubiquity and abundance place peroxiredoxins, along with glutathione peroxidases, as obligate hydroperoxide cellular sensors.

Swapping FAD binding motifs between plastidic and bacterial ferredoxin-NADP(H) reductases.

Plant-type ferredoxin-NADP(H) reductases (FNRs) are grouped in two classes, plastidic with an extended FAD conformation and high catalytic rates and bacterial with a folded flavin nucleotide and low turnover rates. The 112-123 ß-hairpin from a plastidic FNR and the carboxy-terminal tryptophan of a bacterial FNR, suggested to be responsible for the FAD differential conformation, were mutually exchanged. The plastidic FNR lacking the ß-hairpin was unable to fold properly. An extra tryptophan at the carboxy terminus, emulating the bacterial FNR, resulted in an enzyme with decreased affinity for FAD and reduced diaphorase and ferredoxin-dependent cytochrome c reductase activities. The insertion of the ß-hairpin into the corresponding position of the bacterial FNR increased FAD affinity but did not affect its catalytic properties. The same insertion with simultaneous deletion of the carboxy-terminal tryptophan produced a bacterial chimera emulating the plastidic architecture with an increased k(cat) and an increased catalytic efficiency for the diaphorase activity and a decrease in the enzyme's ability to react with its substrates ferredoxin and flavodoxin. Crystallographic structures of the chimeras showed no significant changes in their overall structure, although alterations in the FAD conformations were observed. Plastidic and bacterial FNRs thus reveal differential effects of key structural elements. While the 112-123 ß-hairpin modulates the catalytic efficiency of plastidic FNR, it seems not to affect the bacterial FNR behavior, which instead can be improved by the loss of the C-terminal tryptophan. This report highlights the role of the FAD moiety conformation and the structural determinants involved in stabilizing it, ultimately modulating the functional output of FNRs.

Distance-dependent diffusion-controlled reaction of •NO and O2•- at chemical equilibrium with ONOO-.

The fast reaction of (•)NO and O(2)(•-) to give ONOO(-) has been extensively studied at irreversible conditions, but the reasons for the wide variations in observed forward rate constants (3.8 ≤ k(f) ≤ 20 × 10(9) M(-1) s(-1)) remain unexplained. We characterized the diffusion-dependent aqueous (pH > 12) chemical equilibrium of the form (•)NO + O(2)(•-) = ONOO(-) with respect to its dependence on temperature, viscosity, and [ONOO(-)](eq) by determining [ONOO(-)](eq) and [(•)NO](eq). The equilibrium forward reaction rate constant (k(f)(eq)) has negative activation energy, in contrast to that found under irreversible conditions. In contradiction to the law of mass action, we demonstrate that the equilibrium constant depends on ONOO(-) concentration. Therefore, a wide range of k(f)(eq) values could be derived (7.5-21 × 10(9) M(-1) s(-1)). Of general interest, the variations in k(f) can thus be explained by its dependence on the distance between ONOO(-) particles (sites of generation of (•)NO and O(2)(•-)).

Structural and enzymatic insights into the ATP binding and autophosphorylation mechanism of a sensor histidine kinase.

DesK is a sensor histidine kinase (HK) that allows Bacillus subtilis to respond to cold shock, triggering the adaptation of membrane fluidity via transcriptional control of a fatty acid desaturase. It belongs to the HK family HPK7, which includes the nitrogen metabolism regulators NarX/Q and the antibiotic sensor LiaS among other important sensor kinases. Structural information on different HK families is still scarce and several questions remain, particularly concerning the molecular features that determine HK specificity during its catalytic autophosphorylation and subsequent response-regulator phosphotransfer reactions. To analyze the ATP-binding features of HPK7 HKs and dissect their mechanism of autophosphorylation at the molecular level, we have studied DesK in complex with ATP using high resolution structural approaches in combination with biochemical studies. We report the first crystal structure of an HK in complex with its natural nucleotidic substrate. The general fold of the ATP-binding domain of DesK is conserved, compared with well studied members of other families. Yet, DesK displays a far more compact structure at the ATP-binding pocket: the ATP lid loop is much shorter with no secondary structural organization and becomes ordered upon ATP loading. Sequence conservation mapping onto the molecular surface, semi-flexible protein-protein docking simulations, and structure-based point mutagenesis allow us to propose a specific domain-domain geometry during autophosphorylation catalysis. Supporting our hypotheses, we have been able to trap an autophosphorylating intermediate state, by protein engineering at the predicted domain-domain interaction surface.

Thiol and sulfenic acid oxidation of AhpE, the one-cysteine peroxiredoxin from Mycobacterium tuberculosis: kinetics, acidity constants, and conformational dynamics.

Drug resistance and virulence of Mycobacterium tuberculosis are partially related to the pathogen's antioxidant systems. Peroxide detoxification in this bacterium is achieved by the heme-containing catalase peroxidase and different two-cysteine peroxiredoxins. M. tuberculosis genome also codifies for a putative one-cysteine peroxiredoxin, alkyl hydroperoxide reductase E (MtAhpE). Its expression was previously demonstrated at a transcriptional level, and the crystallographic structure of the recombinant protein was resolved under reduced and oxidized states. Herein, we report that the conformation of MtAhpE changed depending on its single cysteine redox state, as reflected by different tryptophan fluorescence properties and changes in quaternary structure. Dynamics of fluorescence changes, complemented by competition kinetic assays, were used to perform protein functional studies. MtAhpE reduced peroxynitrite 2 orders of magnitude faster than hydrogen peroxide (1.9 x 10(7) M(-1) s(-1) vs 8.2 x 10(4) M(-1) s(-1) at pH 7.4 and 25 degrees C, respectively). The latter also caused cysteine overoxidation to sulfinic acid, but at much slower rate constant (40 M(-1) s(-1)). The pK(a) of the thiol in the reduced enzyme was 5.2, more than one unit lower than that of the sulfenic acid in the oxidized enzyme. The pH profile of hydrogen peroxide-mediated thiol and sulfenic acid oxidations indicated thiolate and sulfenate as the reacting species. The formation of sulfenic acid as well as the catalytic peroxidase activity of MtAhpE was demonstrated using the artificial reducing substrate thionitrobenzoate. Taken together, our results indicate that MtAhpE is a relevant component in the antioxidant repertoire of M. tuberculosis probably involved in peroxide and specially peroxynitrite detoxification.

Sulfenic acid--a key intermediate in albumin thiol oxidation.

The single thiol of human serum albumin (HSA-SH) is the predominant plasma thiol. Both circulating albumin and pharmaceutical preparations are heterogeneous regarding the thiol redox status, as revealed by anion-exchange-hydrophobic interaction chromatography. Sulfenic acid (HSA-SOH) is an intermediate in HSA-SH oxidation processes that was detected through different techniques including mass spectrometry. Recently, quantitative data led to the determination of rate constants. The preferred fate of HSA-SOH is the formation of mixed disulfides. Alternatively, HSA-SOH can be further oxidized to sulfinic and sulfonic acids. Oxidized forms increase under disease conditions, underscoring the importance of HSA-SH as a plasma scavenger of intravascular oxidants. We here provide a critical review of the oxidation of HSA-SH in the context of the intravascular compartment, with emphasis in the methodological approaches of mass spectrometry and chromatography for the analysis of albumin thiol redox states.

Discovery of novel inhibitors of Trypanosoma cruzi trans-sialidase from in silico screening.

trans-Sialidase from Trypanosoma cruzi (TcTS) has emerged as a potential drug target for treatment of Chagas disease. Here, we report the results of virtual screening for the discovery of novel TcTS inhibitors, which targeted both the sialic acid and sialic acid acceptor sites of this enzyme. A library prepared from the Evotec database of commercially available compounds was screened using the molecular docking program GOLD, following the application of drug-likeness filters. Twenty-three compounds selected from the top-scoring ligands were purchased and assayed using a fluorimetric assay. Novel inhibitor scaffolds, with IC(50) values in the submillimolar range were discovered. The 3-benzothiazol-2-yl-4-phenyl-but-3-enoic acid scaffold was studied in more detail, and TcTS inhibition was confirmed by an alternative sialic acid transfer assay. Attempts to obtain crystal structures of these compounds with TcTS proved unsuccessful but provided evidence of ligand binding at the active site.

Reactivity of sulfenic acid in human serum albumin.

Sulfenic acid is formed upon oxidation of thiols and is a central intermediate in the redox modulation of an increasing number of proteins. Methods for quantifying or even detecting sulfenic acid are scarce. Herein, the reagent 7-chloro-4-nitrobenz-2-oxa-1,3-diazole was determined not to be suitable as a chromophoric probe for sulfenic acid in human serum albumin (HSA-SOH) because of lack of specificity. Thionitrobenzoate (TNB) reacted with HSA exposed to hydrogen peroxide, but not control or thiol-blocked HSA. The reaction was biphasic. The first phase was approximately 20-fold faster than the second phase and first order in HSA-SOH and TNB (105 +/- 11 M-1 s-1, 25 degrees C, pH 7.4), allowing quantitative data on HSA-SOH formation and reactivity to be obtained. Exposure of reduced HSA (0.5 mM) to hydrogen peroxide (4 mM, 37 degrees C, 4 min) yielded 0.18 +/- 0.02 mol of HSA-SOH per mol of HSA. HSA-SH reacted with hydrogen peroxide at 2.7 +/- 0.7 M-1 s-1 (37 degrees C, pH 7.4), while HSA-SOH reacted at 0.4 +/- 0.2 M-1 s-1, yielding sulfinic acid (HSA-SO2H), as detected by mass spectrometry. The rate constants of HSA-SOH with targets of analytical interest such as dimedone and sodium arsenite were determined. HSA-SOH did not react appreciably with the plasma reductants ascorbate or urate, nor with free basic amino acids. In contrast, HSA-SOH reacted rapidly with the plasma thiols cysteine, glutathione, homocysteine, and cysteinylglycine at 21.6 +/- 0.2, 2.9 +/- 0.5, 9.3 +/- 0.9, and 55 +/- 3 M-1 s-1 (25 degrees C, pH 7.4), respectively, supporting a role for HSA-SOH in the formation of mixed disulfides.

Design, synthesis, and biological characterization of potential antiatherogenic nitric oxide releasing tocopherol analogs.

Synthesis and biological characterization of a series of alpha-tocopherol analogs with NO-releasing capacity are reported. The selected NO-donor moieties were nitrooxy and furoxan. All products were tested for their in vitro NO-releasing capacities, vasodilating properties, and antiplatelet activity. They were also capable of preventing LDL oxidation.

Reactivity of peroxynitrite and nitric oxide with LDL.

Low density lipoprotein (LDL) oxidation by peroxynitrite is a complex process, finely modulated by control of peroxynitrite formation, LDL availability and free-radical scavenging by nitric oxide (*NO), ascorbate and alpha-tocopherol (alpha -TOH). In the presence of CO2, lipid targets are spared at the expense of surface constituents. Since surface damage may lead to oxidation-induced LDL aggregation and particle recognition by scavenger receptors, CO2 cannot be considered an inhibitor of peroxynitrite-dependent LDL modifications. Chromanols, urate and ascorbate cannot scavenge peroxynitrite in the vasculature, although intermediates of urate oxidation and high ascorbate concentrations may do soin vitro. Most if not all of the protection against peroxynitrite-induced LDL oxidation afforded by urate, ascorbate, chromanols and also*NO should be considered to depend on their free radical scavenging abilities, including inactivation of lipid peroxyl radicals (LOO),*NO2, and CO3*-; as well as their capacity to reduce high oxidation states of metal centers. Peroxynitrite direct interception by reduced manganese (II) porphyrins is possibly the most powerful although unspecific strategy to inhibit peroxynitrite reactions. In light of the recent demonstration of nitrated bioactive lipids in vivo, renewed interest in the mechanisms of peroxynitrite- and nitric oxide-mediated lipid nitration and nitrosation is guaranteed.

Direct measurement of nitric oxide and oxygen partitioning into liposomes and low density lipoprotein.

Nitric oxide (*NO) has been proposed to play a relevant role in modulating oxidative reactions in lipophilic media like biomembranes and lipoproteins. Two factors that will regulate *NO reactivity in the lipid milieu are its diffusion and solubility, but there is no data concerning the actual diffusion (D) and partition coefficients (KP) of *NO in biologically relevant hydrophobic phases. Herein, a "equilibrium-shift" method was designed to directly determine the *NO and O2 partition coefficients in liposomes and low density lipoprotein (LDL) relative to water. It was found that *NO partitions 4.4- and 3.4-fold in liposomes and LDL, respectively, whereas O2 behaves similarly with values of 3.9 and 2.9, respectively. In addition, actual diffusion coefficients in these hydrophobic phases were determined using fluorescence quenching and found that *NO diffuses approximately 2 times slower than O2 in the core of LDL and 12 times slower than in buffer (DNOLDL=3.9 x 10(-6) cm2 s(-1),DO2LDL=7.0 x 10(-6) cm2 s(-1),DNObuffer=DO2buffer=4.5 x 10(-5) cm2 s(-1)). The influence of *NO and O2 partitioning and diffusion in membranes and lipoproteins on *NO reaction with lipid radicals and auto-oxidation is discussed. Particularly, the 3-4-fold increase in O2 and *NO concentration within biological hydrophobic phases provides quantitative support for the idea of an accelerated auto-oxidation of *NO in lipid-containing structures, turning them into sites of enhanced local production of oxidant and nitrosating species.